The long-term goal is to determine the mechanism of neoplastic transformation by tryosine-specific protein kinases. Our approach is to use a membrane-enveloped virus, vesicular stomatitis virus (VSV), as a probe to investigate a subpopulation of tyrosyl kinases in cells. VSV was chosen as a probe since, during virus maturation, tumor antigens as well as tyrosine-specific protein kinases are specifically incorporated into VSV virions. Furthermore, VSV has been shown by us to incorporate elevated levels of tyrosyl kinases from anchorage-independent cells. Thus, our working hypothesis is that tyrosine kinases are relocalized to VSV budding sites in anchorage-independent cells and that this subpopulation of tyrosyl kinases functions in anchorage-independent growth of cells. Therefore, VSV will be used to investigate this subpopulation of tyrosyl kinases in a variety of anchorage-independent cells. During the first year of this project, we have investigated a variety of cell types and directly correlated their anchorage-independent growth with amplified tyrosyl kinase levels in VSV grown in the cells. Further, we have investigated the identity of the tyrosyl kinases that are acquired from one cell line--baby hamster kidney (BHK) suspension cultures which are not transformed by retroviruses but are anchorage-independent for growth. Partial purifications of VSV tyrosyl kinases and cellular tyrosyl kinases from BHK suspension cells show that one specific tyrosyl kinase is acquired by VSV. The tyrosyl kinase is unrelated to pp60src, and it does not respond to growth factors, indicating that the kinase acquired by VSV is not a growth factor receptor. In the final phase of our project, we will characterize and localize in cells the VSV-acquired tyrosyl kinase. To do this, large quantities of the VSV-associated tyrosyl kinase will be purified for molecular weight determinations for testing with monoclonal anti-serum, which cross reacts with several oncogene tyrosyl kinases, and for antibody production. When antibodies are obtained, they will be used for immunofluorescence localization in anchorage-dependent and -independent cells. We also will use VSV to investigate the tyrosyl kinases in cells transformed by retroviruses and DNA tumor viruses that do not encode tyrosyl kinases. (B)